The invention relates to a specific class of single-chain fertility hormones. The single-chain forms contain two xcex2 subunits upstream of the a subunit. The hormones of the invention have similar activities with respect to follicle stimulating hormone (FSH) function, but differing activities with respect to luteinizing hormone (LH) function. Thus, the compounds of the invention have variable ratios of FSH and LH activity.
In humans, four important glycoprotein hormone heterodimers (LH, FSH, thyroid stimulating hormone (TSH) and chorionic gonadotropin CG) have identical xcex1 subunits and differing xcex2 subunits. Three of these hormones are present in virtually all other vertebrate species as well; CG has so far been found only in primates and in the placenta and urine of pregnant mares. FSH is an important hormone in regulating fertility; FSH has been used both in vivo and in vitro to enhance fertility. LH also appears to play a role in such treatments.
Two published PCT applications describe single chain forms of the four hormones FSH, LH, TSH and CG wherein the xcex1 and xcex2 unit are covalently linked to result in a fusion peptide of the general formulas:
xcex2(linker)nxcex1 or
xcex1(linker)nxcex2
wherein n is 0 or 1 and xcex1 and xcex2 represent the respective subunits of these hormones: Moyle, W. R., PCT application WO95/22340 published Aug. 24, 1995 and the application of the inventor herein, WO96/05224 published Feb. 22, 1996. The disclosure of these documents is incorporated herein by reference.
Forms of the above-described single-chain glycoprotein hormones in which the number of cystine bridges has been depleted are disclosed in U.S. Ser. No. 08/933,693 filed Sep. 19, 1997, and incorporated herein by reference.
PCT application published as WO99/25849, published May 27, 1999, discloses additional single-chain forms which contain two xcex2 subunits linked to a single xcex1 subunit. These proteins are of the formula:
xcex21 (linker1)m-xcex22-(linker2)n-xcex1;
xcex1-(linker1))m-xcex21-(linker1))n-B2 and
xcex21-(linker1))m-xcex1-xcex22-(linker2))n.
This genus includes a large number of individual members and the publication does not focus on any particular subclass. The contents of this document are incorporated herein by reference.
In addition, PCT application PCT/US 99/23555 filed Oct. 12, 1999 and also incorporated herein by reference, discloses additional forms of the hormones which contain two xcex2 subunits, but wherein one of the xcex2 subunits is bound non-covalently to a single-chain form containing a single xcex2 and an xcex1 subunit.
Of additional relevance to present invention are publications wherein the carboxy terminal peptide (CTP) of human chorionic gonadotropin or a variant thereof is used to modify pharmaceuticals in general, and these hormones in particular. Thus, PCT application publication number WO90/09800, published Sep. 7, 1990, and incorporated herein by reference, describes various modified forms of these hormones, including C-terminal extension of the xcex2 subunit by CTP or a variant. Other muteins of these hormones are also described. xe2x80x9cCTPxe2x80x9d is the sequence of amino acids extending from any one of positions 112-118 to position 145 of the xcex2 subunit of human chorionic gonadotropin. In addition, PCT application publication number WO94/24148 published Oct. 27, 1994, incorporated herein by reference, describes modifying these hormones and other compounds by extension or insertion of the CTP at locations other than the C-terminus and with CTP fragments shorter than the sequence extending from positions 112-118 to 145.
The CTP-extended xcex2 subunit of FSH is also described in two papers by applicants herein: LaPolt, P. S., et al., Endocriniology (1992) 131:2514-2520 and Fares, F. A., et al., Proc Natl Acad Sci USA (1992) 89:4304-4308. Both of these papers are incorporated herein by reference. Also incorporated by reference is an article by the inventors herein which describes the activity of a single-chain compound:. FSHxcex2-CG xcex2-xcex1: Masatoshi Kanda, et al., Molecular Endocrinology (1999) 13: 1873-1881.
The crystal structure of the heterodimeric form of human chorionic gonadotropin has now been published in more or less contemporaneous articles; one by Lapthom, A. J., et al., Nature (1994) 369:455-461 and the other by Wu, H., et al., Structure (1994) 2:545-558. The results of these articles are summarized by Patel, D. J., Nature (1994) 369:438-439.
PCT application WO91/16922 published Nov. 14, 1991 describes a multiplicity of chimeric and otherwise modified forms of the heterodimeric glycoprotein hormones. In general, the disclosure is focused on chimeras of xcex1 subunits or xcex2 subunits involving portions of various xcex1 or xcex2 chains respectively. One construct simply listed in WO91/16922, and not otherwise described, fuses substantially all of the xcex2 chain of human chorionic gonadotropin to the xcex1 subunit preprotein, i.e., including the secretory signal sequence for this subunit.
It has now been found that a particular subset of the genus of biofunctional single-chain compounds disclosed in the above referenced PCT publication WO99/25849 have particularly advantageous properties when used in protocols for enhanced fertility in vitro and in vivo. In this subset, FSH activity is maintained at a level comparable to that of the native hormone, but LH activity is varied over a range generally lower than that of native LH.
The invention provides single-chain forms of fertility inducing hormones that have varying ratios of LH activity as compared to FSH activity. In general, the, single-chain forms have FSH agonist activity comparable to the activity exhibited by equivalent amount of native FSH, but have LH agonist activity which is variable among the various embodiments of the class. Generally, the compounds have LH agonist activities in varying degrees, but typically less than that associated with the native hormone. The single-chain forms of the invention may either be glycosylated, partially glycosylated, or nonglycosylated and the FSHxcex2 is linked through a linker to LHxcex2. The xcex1 subunit is downstream from both.
Thus, in one aspect, the invention is directed to a glycosylated or nonglycosylated protein of the formula
FSHxcex2-(linker1)n1-LHxcex2(1-X)-(linker2)n2-xcex1
wherein xe2x80x9cxcex1xe2x80x9d designates the common xcex1 subunit of the glycoprotein hormones or a variant thereof; FSHxcex2 refers to the xcex2 subunit of follicle stimulating hormone; LHxcex2(1-X) refers to the luteinizing hormone xcex2 subunit optionally containing deletion of up to seven amino acids from said carboxy terminus; each xe2x80x9clinkerxe2x80x9d is an amino acid sequence which is flexible and hydrophilic and may be CTP. CTP refers to the carboxy terminal peptide of chorionic gonadotropin xcex2 subunit as further defined hereinbelow. Each n is 0 or 1. However, in one set of preferred embodiments, if both n=1 and both linkers=CTP, X cannot be 114 or if linker1 is CTP, LHxcex2(1-X)-(linker2)- cannot be replaced by CGxcex2.
In other aspects, the invention is directed to recombinant materials and methods to produce the proteins of the invention, to pharmaceutical compositions containing them; to antibodies specific for them; and to methods for their use.